Embryonic stem cells (ESC) are particularly unique in that they process a high degree of plasticity not shared by other stem cells such as hematopoietic or mesenchymal stem cells. More importantly, ESC show a low degree of immunogenicity by expressing little or no MHC class I antigens and no class II antigens even after treatment with IFN-gamma. Little is, however, known about the conditions under which they can optimally induce a state of mixed chimerism that would be critical for their use in organ transplantation, but also in the treatment of degenerative diseases, autoimmune diseases and other illnesses. In organ transplantation, approximately 50% of allografts are lost within 10 years by a process generally termed chronic rejection, indicating the need for the establishment of immunological tolerance. One strategy to achieving tolerance to transplanted organs is by creating a state of mixed-chimerism using donor-derived hematopoietic cells. The severity of the available myelo-ablative protocols necessary for the achievement of mixed chimerism are too severe for patient awaiting solid organ transplantation, but remain the treatment of choice for patients suffering from malignant leukemic diseases. In contrast, most recent data in rats show that donor-type embryonic stem cell-lines are capable of inducing mixed-chimerism and subsequent cardiac graft tolerance without host pre-conditioning or immunosuppression. Here, we propose to test whether mouse embryonic stem cells (ESC) induce mixed-chimerism in allogeneic combinations and tolerance to cardiac allografts without immunosuppression or under minimal host treatment. The central hypothesis is that mouse ESC, which are immune-privileged, establish a state of mixed- chimerism in allogeneic recipients in the absence of pre-conditioning regimens or under minimal host treatment, in a thymus-dependent fashion, and that the development of mixed-chimerism permits permanent engraftment of vascularized cardiac allografts. Aim 1 will establish ideal conditions for achieving ESC-induced mixed-chimerism and determine whether established mixed chimerism induces tolerance to donor-derived vascularized cardiac allografts. Aim 2 will deal with the mechanisms for ESC immune-privilege including determining whether ESC induce clonal deletion of alloreactive T cells. It is anticipated that mixed-chimerism and tolerance in this mouse model will be achieved, using this innovative approach that avoids toxic pre-conditioning regimens. The results of these collective studies will be an important step in understanding the efficacy of ESC to engraft in allogeneic recipients. Eventually, this data could be utilized for the induction of mixed chimerism in humans that is essential for tolerance induction.